ES2375843T3 - PROCEDURES AND COMPOUNDS FOR THE PREPARATION OF ANC? LOGOS OF CC-1065. - Google Patents

Abstract

A manufacturing process of a compound (I) or a salt thereof, wherein R 1 and R 2 are each independently H, substituted alkyl or unsubstituted alkyl, -C (O) OR ', - C (O) NR'R ", or a protecting group, wherein R 'and R" are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, heterocycloalkyl substituted and unsubstituted heterocycloalkyl, R 6 is H, substituted C 1-6 alkyl or unsubstituted C 1-6 alkyl, cyano or alkoxy; and X is halogen, the process comprising: the addition of protective groups R 1 'and R 2' to a compound (II) to form a compound (III) in which R 3 is substituted alkyl or unsubstituted alkyl; and ** Formula ** generate a five-membered ring comprising the amino nitrogen of compound (III) by alkylation of the aryl ring adjacent to the nitrogen followed by ring closure.

Description

Procedures and compounds for the preparation of analogs of CC-1065

Background

It is known that CC-1065 is a potent cytotoxin. CC-1065 was isolated for the first time from Streptomyces zelensis

5 in 1981 by the Upjohn Company (Hanka et al., J. Antibiot. 31: 121 1 (1978); Martin et al., J. Antibiot. 33: 902 (1980); Martin et al., J. Antibiot. 34: 1119 (1981)) and was found to have potent antitumor and antimicrobial activity both in vitro and in experimental animals (Li et al., Cancer Res. 42: 999 (1982)). CC-1065 binds to double-stranded B-DNA within the minor groove (Swenson et al., Cancer Res. 42: 2821 (1982)) with the sequence preference of 5'-d (A / GNTTA) -3 ' and 5 '- (AAAAA) -3' and rents the N3 position of 3'-adenine through its part unit

10 left CPI present in the molecules (Hurley et al, Science 226: 843 (1984)). Despite its potent and extensive antitumor activity, CC-1065 cannot be used in humans because it causes delayed death in experimental animals.

Many analogs and derivatives of CC-1065 are known in the art. The research on structure, synthesis and properties of many of the compounds has been reviewed. See, for example, Boger et al, Angew. Chem. Int. Ed.

15 Engl. 35: 1438 (1996); and Boger et al, Chem. Rev. 97: 787 (1997).

A group of Kyowa Hakko Kogya Co., Ltd. has prepared a number of derivatives of CC-1065. See, for example, U.S. Patents Nos. 5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780; 5,101,038; and 5,084,468; and the published PCT application, WO 96/10405 and the published European application 0 537 575 A1.

20 The Upjohn Company (Pharmacia Upjohn) has also been active in the preparation of derivatives of CC-1065. See, for example, U.S. Patents Nos. 5,739,350; 4,978,757. 5,332. 837 and 4,912,227.

Brief Summary

An embodiment is a process of preparing a compound (1) or a salt thereof,

Wherein R1 and R2 are each independently H, substituted alkyl or unsubstituted alkyl, -C (O) OR ', -C (O) NR'R ", or a protecting group, where R' and R" are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl, R6 is H, substituted C1-6 alkyl or C1- alkyl 6 unsubstituted, cyano or alkoxy; and X is halogen, the process comprising:

30 the addition of protective groups R1 'and R2' to a compound (II)

(II)

forming a compound (III) in which R3 is substituted alkyl or unsubstituted alkyl; Y

a five-membered ring comprising the amino nitrogen of compound (III) is generated by alkylation of the aryl ring adjacent to the nitrogen followed by ring closure.

Another embodiment is a method of preparing the CBI analog CC-1065, or a pharmaceutically acceptable salt thereof, which has the following formula:

10 in which X is halo;

X1 and Z are each independently selected from O, S and NR8, wherein R8 is a member selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, and acyl;

R4, R4 ', R5 and R5' are members independently selected from the group consisting of H, substituted alkyl,

15 unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO2, NR9R10, NC (O) R9, OC (O) NR9R10, OC (O) OR9, C (O) R9, SR9, OR9 and CR9 = NR10,

wherein R9 and R10 are independently selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, substituted aryl or unsubstituted aryl, heteroaryl

Substituted or unsubstituted heteroaryl, substituted heterocycloalkyl or unsubstituted heterocycloalkyl, and substituted peptidyl or unsubstituted peptidyl or those R9 and R10 together with the nitrogen atom to which they are attached are optionally combined to form a substituted or unsubstituted heterocycloalkyl ring system which has 4 to 6 members, optionally containing two or more heteroatoms, and

R11 and R11 'are each independently H or substituted C1-6 alkyl or unsubstituted C1-6 alkyl;

R6 is H, substituted C1-6 alkyl or unsubstituted C1-6 alkyl, cyano, or alkoxy, and

R7 is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C (O) R12R13, C (O) OR12, C (O) NR12R13, P (O) (OR12) 2, C (O) CHR12R13, SR12 and SiR12R13R14,

where R12, R13, and R14 are independently selected members of H, substituted alkyl or non-alkyl

30 substituted, substituted heteroalkyl or unsubstituted heteroalkyl and substituted aryl or unsubstituted aryl, or where R12 and R13 together with the nitrogen or carbon atom to which they are attached are optionally combined forming a substituted or unsubstituted heterocycloalkyl ring system having 4 to 6 members, optionally containing two or more heteroatoms; Understanding the procedure:

the addition of protective groups R1 'and R2' to a compound (II)

forming a compound (III)

wherein R3 is substituted alkyl or unsubstituted alkyl;

generating a five-membered ring comprising the amino nitrogen of compound (III) by alkylation of the aryl ring adjacent to the nitrogen followed by ring closure; and adding a binding unit to compound (III) by removing the protective group R1 ’and joining the rest:

to the amino substituent of compound (III), in which indicates the point at which the residue is attached to the amino substituent.

A compound of formula (I), or a pharmaceutically acceptable salt thereof, is also disclosed:

wherein R1 and R2 are each independently H, alkyl, -C (O) OR ', -C (O) NR'R' 'or a protecting group, where R' and R "are independently selected from the group consisting in H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl.

Brief description of the drawings

Non-limiting and non-exhaustive embodiments of the present invention are described with reference to the following drawings. In the drawings, the same numerical references refer to parts of the various figures unless otherwise specified.

For a better understanding of the present invention, the reference will be made to the following detailed description that must be contemplated together with the accompanying drawings, in which

FIG. 1 is a synthesis scheme of an embodiment of a method of forming a CBI analogue CC-1065;

FIG. 2 is a synthesis scheme of another embodiment of a method of forming a CBI analogue CC-1065; Y

FIG. 3 is a synthesis scheme of a third embodiment of a method for forming a CBI analogue CC-1065.

Detailed description

As used in this invention, "Boc" refers to t-butyloxycarbonyl.

"CPI" refers to cyclopropapyrrolindole.

"CBI" refers to cyclopropabencindol.

"Cbz" is carbobenzoxy.

"DCM," refers to dichloromethane.

"DMF" is N, N-dimethylformamide.

"FMOC" refers to 9-fluorenylmethyloxycarbonyl.

"ASD" refers to triethylamine.

"THF" refers to tetrahydrofuran.

"EDC" refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

Unless defined otherwise, all the technical and scientific terms used in this invention generally have the same meaning that is usually understood by a person skilled in the art to which this invention belongs. In general, the nomenclature used in this invention and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below is well known and is commonly used in the art. The techniques and procedures are generally carried out in accordance with conventional procedures in the art and various general references. The nomenclature used in this invention and the laboratory procedures in analytical chemistry and organic synthesis described below are well known and commonly used in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis.

The term "therapeutic agent" is intended to mean a compound that when present in a therapeutically effective amount, produces a desired therapeutic effect in a mammal. For the treatment of carcinomas it is desirable that the therapeutic agent is also able to enter the target cell.

The term "cytotoxin" is intended to mean a therapeutic agent that has the desired effect of being cytotoxic for cancer cells. Cytotoxic means that the agent counteracts the growth of, or kills, the cells.

The terms "prodrug" and "drug conjugate" are used interchangeably in this invention. Both refer to a compound that is relatively harmless to cells even in the conjugated form but that is selectively degraded in a pharmacologically active form by conditions, for example, enzymes, located within or in the vicinity of target cells.

The symbol

 when used as a bond or shown perpendicular to a bond, it indicates the point at which the rest shown is attached to the rest of the molecule, solid support, substituent, etc.

The term "alkyl" by itself or as part of another substituent, means, unless otherwise established, a linear or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be completely saturated, mono - or polyunsaturated and may include di- and multivalent radicals, having the designated number of carbon atoms (ie, C1-C10 means one to ten carbon). Examples of radicals

saturated hydrocarbons include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one that has one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1 - and 3-propyl, 3-butynyl, and higher homologs and isomers. The term "alkyl", unless otherwise indicated, is also understood to include those alkyl derivatives defined in more detail below, such as "heteroalkyl." Alkyl groups, which are limited to hydrocarbon groups are called "homoalkyl."

The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited to, with -CH2CH2CH2CH2-, and also includes those groups described below as "heteroalkylene". Typically an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, those groups having 10 or less carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, which generally has eight or less carbon atoms.

The term "heteroalkyl" by itself or in combination with another term, means, unless otherwise established, a stable linear or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of a number of atoms. of carbon and at least one heteroatom selected from the group consisting of O, N, Si and S, and in which the nitrogen, carbon and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom (s) O, N, S and Si can be arranged in any interior position of the heteroalkyl group or in the position in which the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2 -CH2, -S (O) -CH3, -CH2-CH2-S (O) 2-CH3, -CH = CH-O-CH3, -Si (CH3) 3, -CH2-CH = N-OCH3, and -CH = CH-N (CH3) -CH3. Up to two heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si (CH3) 3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited to, with -CH2-CH2-S-CH2-CH2- and -CH2 -S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the heteroatoms can also occupy one or both of the chain terminals (for example, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamine, and the like). The terms "heteroalkyl" and "heteroalkylene" comprise poly (ethylene glycol) and its derivatives (see, for example, Shearwater Polymers Catalog, 2001). In addition to alkylene and heteroalkylene substituents no orientation of the substituent is implied with the direction in which the substituent formula is written. For example, the formula -C (O) 2R'- represents both -C (O) 2R'- and - R’C (O) 2-.

The term "lower" in combination with the terms "alkyl" or "heteroalkyl" refers to a moiety having 1 to 6 carbon atoms.

The terms "alkoxy", "alkylamino", "alkylsulfonyl" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the rest of the molecule by an oxygen atom, an amino group , a SO2 group or a sulfur atom, respectively. The term "arylsulfonyl" refers to an aryl group attached to the rest of the molecule by an SO2 group, and the term "sulfhydryl" refers to a SH group.

In general, an "acyl" substituent is also selected from the group described below. As used herein, the term "acyl" substituent refers to groups attached to, and fulfills the valence of a carbonyl carbon that is directly or indirectly attached to the polycyclic core of the compounds of the present invention.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise indicated, cyclic versions of "substituted or unsubstituted alkyl" and "substituted or unsubstituted heteroalkyl," respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position in which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran -3-yl, tetrahydrothien-2-yl, tetrahydrothien-3yl, 1-piperazinyl, 2-piperazinyl, and the like. The heteroatoms and carbon atoms of the cyclic structures are optionally oxidized.

The terms "halo" or "halogen", by themselves or as part of other substituents, means, unless otherwise indicated, a fluorine, chlorine, bromine or iodine atom. Additionally, terms such as "haloalkyl" are understood to include monoalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is understood to include, but not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term "aryl" means, unless otherwise indicated, a hydrocarbon substituent, substituted or unsubstituted, polyunsaturated, aromatic, which may be a single ring or multiple rings (preferably 1 to 3 rings) that are condensed together or united covalently. The term "heteroaryl" refers to aryl groups (or rings) containing one to four "heteroatoms selected from N, O, and S, in which the nitrogen, carbon and sulfur atoms are optionally oxidized, and the (the ) nitrogen atom (s) are

optionally quaternized. A heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl , 4-oxazolyl, 2-phenyl-4oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3 -thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl , 3-quinolyl, and 6-quinolyl. Substituents for each of the aryl and heteroaryl ring systems indicated above are selected from the group of acceptable substituents described below. "Aryl" and "heteroaryl" also comprise ring systems in which one or more non-aromatic ring systems are condensed, or otherwise bound, to an aryl or heteroaryl system.

By way of summary the term "aryl" when used in combination with other terms (for example, aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is understood to include those radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which an atom has been replaced. carbon (for example, a methylene group), for example, with an oxygen atom (for example, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like).

Each of the above terms (for example, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) are generally referred to as "alkyl substituents" and "heteroalkyl substituents," respectively. , and may be one or more of a variety of groups selected from, but not limited to: -OR ', = O, = NR', = N-OR ', -NR'R ", -SR', -halogen , -SiR'R "R '", -OC (O) R', -C (O) R ', -CO2R', -CONR'R ", -OC (O) NR'R", NR''C (O) R ', -NR'-C (O) NR "R"', -NR "C (O) 2R ', NR-C (NR'R" R' ") = NR" ", -NR- C (NR'R ") = NR" ', -S (O) R', -S (O) 2R ', -S (O) 2NR'R ", -NRSO2R', -CN and -NO2 in a number which varies from zero to (2m '+ 1), where m' is the total number of carbon atoms in such a radical. R ', R ", R"' and R "" each preferably independently refers to hydrogen, heteroalkyl substituted or unsubstituted, aryl substituted or not substituted, for example, aryl substituted with 1 to 3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one group R, for example, each of the groups R is independently selected since they are each groups R ', R ", R'" and R "" when more than one of these Groups is present. When R 'and R "are attached to the same nitrogen atom forming these they can be combined with the nitrogen atom forming a 5, 6 or 7 member ring. For example, it is understood that -NR'R" includes, but is not limited to to these, 1-pyrrolidinyl and 4-morpholinyl. From the above description of substituents, a person skilled in the art will understand that the term "alkyl" is understood to include groups that include carbon atoms attached to groups other than hydrogen groups, such as haloalkyl (for example, -CF3 and -CH2CF3) and acyl (for example, C (O) CH3, -C (O) CF3, -C (O) CH2OCH3, and the like).

Similar to the substituents described for the alkyl radical, aryl substituents and heteroaryl substituents are generally referred to as "aryl substituents" and "heteroaryl substituents" respectively and vary and are selected, for example, from: halogen, -OR ', = O, = NR '= N-OR', -NR'R ", -SR ', -halogen, -SiR'R" R "', -OC (O) R ', -C (O) R', -CO2R ', -CONR'R ", -OC (O) NR'R", -NR "C (O) R', -NR'-C (O) NR" R '", -NR" C (O ) 2R ', -NR-C (NR'R ") = NR"', -S (O) R ', -S (O) 2R', -S (O) 2NR'R ", -NRSO2R ', - CN and -NO2, -R ', -N3, -CH (Ph) 2, fluoroalkoxy (C1-C4) and fluoroalkyl (C1-C4), in a number that varies from zero to the total number of open valences in the system aromatic ring; and where R ', R ", R'" and R "" are preferably independently selected from hydrogen, (C1-C8) alkyl and unsubstituted heteroalkyl, aryl and heteroaryl, (unsubstituted aryl) -C1-C4 alkyl, and (unsubstituted aryl) oxy-(C1-C4) alkyl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected since they are each R ', R ", R'" and R "" groups, when more than one of These groups are present.

Two of the aryl substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced with a substituent of the formula -TC (O) - (CRR ') qU-, in which T and U are independently -NR-, -O -, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced with a substituent of formula -A- (CH2) rB-, in which A and B are independently -CRR'-, -O-, -NR-, -S-, -S (O) -, -S (O) 2-, -S (O ) 2NR '

or a single bond, and r is an integer from 1 to 4. One of the simple bonds of the new ring thus formed may optionally be replaced with a double bond. Alternatively two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of formula - (CRR ') sX- (CR "R'") d-, where syd are independently integers of 0 a 3, and X is -O-, -NR'-, -S-, -S (O) -, -S (O) 2-or -S (O) 2NR'-. The substituents R, R ', R "and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6) alkyl.

As used in this invention, the term "diphosphate" includes, but is not limited to, a phosphoric acid ester containing two phosphate groups. The term "triphosphate" includes, but is not limited to, an acid ester

phosphoric containing three phosphate groups. For example, particular drugs that have a diphosphate or a triphosphate include:

As used in this invention, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

The symbol "R" is a general abbreviation representing a substituent group that is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted heteroaryl

or unsubstituted, and substituted or unsubstituted heterocyclyl groups.

With regard to the term "protecting group", those skilled in the art will understand how to protect a particular functional group from interference with a selected set of reaction conditions. For example, of useful protecting groups, see Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991. Examples of suitable protecting groups include, but are not limited to, BOC, FMOC, 2-trimethylsilylethoxycarbonyl , allyloxycarbonyl, 4-methyl-1-piperazincarbonyl, 1-methyl-1- (4-biphenylyl) ethoxycarbonyl, diphenyloxycarbonyl, benzyl, t-butyl, tetrahydropyran, trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyl, t-butyldiphenyl, t-butyldifyl 2-Trichloroethyloxycarbonyl, diisopropylmethyloxycarbonyl, vinyloxycarbonyl, methoxybenzyloxycarbonyl, nitrobenzyloxycarbonyl, cyclohexyloxycarbonyl, cyclopentyloxycarbonyl, benzyloxycarbonyl, formyl, acetyl, trihaloacetyl, benzoyl, nitrophenylacetyl dithioacetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl acetyl 2-trichloroxycarbonyl ester

The term "pharmaceutically acceptable carrier", as used in this invention, means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulation material, involved in carrying out or transport a chemical agent Pharmaceutically acceptable carriers include pharmaceutically acceptable salts, where the term "pharmaceutically acceptable salts" includes salts of the active compounds that are prepared with relatively non-toxic acids or bases, depending on the particular substituents found in the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either by mass or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic or magnesium amino salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either by mass or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogen phosphoric, dihydrogen phosphoric, sulfuric, monohydrogen sulfuric, iohydric, or phosphorous and the like, as well as salts derived from acids. relatively non-toxic organic such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galacturonic acids and the like (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1 -19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be transformed into base or acid addition salts.

The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise they are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds that are in a prodrug form. The prodrugs of the compounds described in this invention are those compounds that readily undergo chemical changes under physiological conditions by providing the compounds of the present invention. Additionally, prodrugs can be transformed into the compounds of the present invention by chemical or biochemical procedures in an ex vivo environment. For example, prodrugs can be slowly transformed into compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention may exist in non-solvated forms as well as in solvated forms, including hydrated forms. In general, solvated forms are equivalent to non-solvated forms and are within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds, racemates, diastereomers, geometric isomers and individual isomers are within the scope of the present invention.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes in one or more of the atoms constituting such compounds. For example, the compounds may be radiologically labeled with radioactive isotopes such as, for example, tritium (3H), iodine-125 (125I) or carbon14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be within the scope of the present invention.

The terms "polypeptide", "peptide" and "protein" are used interchangeably in this invention to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a naturally occurring amino acid, as well as amino acid polymers of unnatural origin. These terms also include the term "antibiotic."

The term "amino acid" refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded with the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is a carbon to which it is attached to a hydrogen, a carboxyl group, an amino group and an R group, for example, homoserine, norleucine, methionine sulfoxide, methioninemethylsulfonium. Such analogs have modified R groups (eg norleucine) or modified peptide skeletons, but retain the same basic chemical structure as a naturally occurring amino acid. One amino acid that can be used in particular is citrulline, which is an arginine precursor and is involved in the formation of urea in the liver. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but works in a similar way to a naturally occurring amino acid. The term "unnatural amino acid" is intended to represent the "D" stereochemical form of the twenty naturally occurring amino acids described above. It is further understood that the term "unnatural amino acid" includes homologues of natural amino acids and synthetically modified forms of natural amino acids. Synthetically modified forms include, but are not limited to, amino acids having cut or elongated alkylene chains up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprising halogenated groups, preferably halogenated alkyl and aryl groups. When attached to a connector or conjugate of the invention, the amino acid is in the form of an "amino acid side chain," where the amino acid carboxylic group has been replaced with a keto (C (O)) group. Therefore, for example, an alanine side chain is -C (O) -CH (NH2) -CH3, and so on.

Amino acids and peptides can be protected by blocking groups. A blocking group is an atom or a chemical moiety that protects the N-terminus of an amino acid or a peptide from unwanted reactions and can be used during the synthesis of a drug-cleavable substrate conjugate. It should remain attached to the term N throughout the synthesis, and can be eliminated after completing the synthesis of the drug conjugate by chemical conditions or other conditions that selectively achieve its elimination. Suitable blocking groups for the protection of the term N are well known in the art of peptide chemistry. Examples of blocking groups include, but are not limited to, hydrogen, D-amino acid, and carbobenzoxy chloride (Cbz).

The term "antibody" as defined in this invention includes complete antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains CH1, CH2 and CH3, and can be of the mu, delta, gamma, alpha or epsilon isotype. Each light chain is comprised of a variable light chain region (VL) and a constant light chain region. The light chain constant region is comprised of a domain, CL, which can be of the kappa or lambda isotype. The VH and VL regions can be further subdivided into regions of hypervariability, the so-called complementarity determining regions (CDR), interspaced with regions that are more conserved regions, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain a binding domain that interacts with an antigen. Constant regions of the antibodies may mediate the binding of immunoglobulin with tissues or host factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

The terms "antibody fragment" or "antigen binding portion" of an antibody (or simply "antibody portion"), as used in this invention, refers to one or more fragments of an antibody that retain the ability of specifically binding to an antigen. It has been shown that the antigen binding function of an antibody can be developed by fragments of a full length antibody. Examples of binding fragments comprised within the term "antibody fragment" or "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CH1 domains;

(ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the anchor region; (iii) an Fd fragment comprising the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546), which comprises a VH domain ; and (vi) an isolated complementarity determining region (CDR). Additionally, although the two domains of the Fv fragment, VL and VH, are encoded for separate genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be prepared as a simple protein chain in which the pair of VL and VH regions form monovalent molecules (known as single chain Fv (scFv); see for example, Bird et al. (1988) Science 242: 423426; and Huston et al. (1988) Proc. Natl. Acad. Set. USA 85: 5879-5883). Such single chain antibodies are also intended to be comprised within the term "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are selected for their usefulness in the same manner as intact antibodies.

The terms "monoclonal antibody" as used in this invention refer to a preparation of antibody molecules of simple molecular composition. A monoclonal antibody composition shows a specific binding affinity and specificity for a given epitope.

"Solid support", as used in this invention, refers to a material that is substantially insoluble in a selected solvent system, or that can be easily separated (for example, by precipitation) from a selected solvent system in which it is soluble. Solid supports useful in the practice of the present invention may include groups that are activated or capable of being activated to allow selected species to bind to the solid support. A solid support can also be a substrate, for example, a chip, wafer or well, on which an individual compound or more than one compound of the invention is used.

The compounds of the invention are prepared as a simple isomer (for example, enantiomer, cis-trans, positional, diastereomer) or as a mixture of isomers. In a preferred embodiment the compounds are prepared substantially as a simple isomer. Methods for preparing substantially isomerically pure compounds are known in the art. For example, enantiomerically enriched mixtures and enantiomerically pure compounds can be prepared using synthetic intermediates that are enantiomerically pure in combination with reactions that leave the stereochemistry in a chiral center uncharged or give rise to their complete inversion. Alternatively, the final product or intermediates along the synthetic route can be resolved in a single stereoisomer. The techniques for inverting or leaving a particular stereocenter unloaded, and those for resolving mixtures of stereoisomers are well known in the art and the ability of a person skilled in the art to select an appropriate procedure for a given situation is well known. See generally Furniss et al. (eds.), VOGEL's ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5th ED., Longman Scientific and Technical Ltd., Essex, 1991, pages 809-816; and Heller, Ace. Chem. Res. 23: 128 (1990).

Cytotoxic analogs of CC-1065 can be formed using a cyclopropabencindol (CBI) moiety as an alkylating unit instead of the cyclopropapyroloindole (CPI) moiety of CC-1065. As an example, analogs of CBI CC1065 include, but are not limited to, compounds having the formula (or a pharmaceutically acceptable salt thereof):

in which X is halo. Preferably, X is Cl or Br and, more preferably, X is Br.

X1 and Z are each independently selected from O, S and NR8 where R8 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl.

R4, R4 ', R5 and R5' are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO2, NR9R10, NC (O) R9, OC (O) NR9R10, OC (O) OR9, C (O) R9, SR9, OR9 and CR9 = NR10,

Where R9 and R10 are independently selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, substituted aryl or unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl, substituted heterocycloalkyl or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl substituted, or when R9 and R10 together with the nitrogen atom to which they are attached are optionally combined to form a substituted or unsubstituted heterocycloalkyl ring system having 4 to 6 members, optionally containing two or more heteroatoms, and

R11 and R11 'are each independently H or substituted C1-6 alkyl or unsubstituted C1-6 alkyl.

R6 is H, substituted C1-6 alkyl or unsubstituted C1-6 alkyl, cyano, or alkoxy. Preferably R6 is methyl, cyano or H. More preferably, R6 is H.

R7 is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C (O) R12R13, C (O) OR12, C (O) NR12R13, P (O) (OR12) 2, C (O) CHR12R13, SR12 and SiR12R13R14, in which R12, R13 and R14 are independently selected members of H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl and substituted aryl or unsubstituted aryl, or where R12 and R13 together with the nitrogen or carbon atom to which they are attached are optionally combined to form a substituted or unsubstituted heterocycloalkyl ring system having 4 to 6 members, optionally containing two or more heteroatoms

Examples of CBI analogs CC-1065 are described in United States patent applications legally assigned serial numbers 10 / 160,972; 10 / 161,233; 10 / 161,234, 11 / 134,685, and 11 / 134,826. These references also describe examples of synthesis and uses for these compounds. These compounds can be used as therapeutic agents (for example, drugs) and as prodrugs. In at least some embodiments, the CBI CC-1065 analogs can be conjugated to targeting agents, such as an antibody, receptor, peptide, lectins, saccharide, nucleic acid or a combination thereof, for use in pharmaceutical compositions that release form Selective cytotoxic CBI CC-1065 analogs in the desired target cells, such as carcinoma cells.

Representative examples of precancerous conditions that can be treated by these compounds, include, but are not limited to: metaplasia, hyperplasia, dysplasia, colorectal polyps, actinic ketatosis, actinic cheilitis, human papillomavirus, leukoplachy, lichen planus and Bowen's disease.

Representative examples of cancers or tumors that can be treated with these compounds include, but are not limited to: lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, cancer of the central nervous system, kidney cancer, kidney cancer, pancreatic cancer, stomach cancer, mouth cancer, nose cancer, cervical cancer and leukemia. It will be readily apparent to the person skilled in the art that the particular indicated agent can be selected such that it directs the drug to the tumor tissue to be treated with the drug (i.e., a specific targeting agent for a specific antigen is chosen of the tumor). Examples of such addressing agents

they are well known in the art, non-limiting examples of these include anti-Her2 for the treatment of breast cancer, anti-CD20 for treatment of lymphoma, anti-PSMA for treatment of prostate cancer and anti-CD30 for treatment of lymphomas, anti-PSMA for the treatment of prostate cancer and anti-CD30 for the treatment of lymphomas, including non-Hodgkin lymphoma.

These compounds provide a procedure to kill a cell. The process includes the administration to the cell of an amount of a compound of the invention sufficient to kill said cell. In an example embodiment, the compound is administered to a subject carrying the cell. In a further exemplary embodiment the administration serves to retard or stop the growth of a tumor that includes the cell (for example, the cell can be a tumor cell). For administration to retard growth, the cell growth rate should be at least 10% lower than the growth rate before administration. Preferably the growth rate will be delayed at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or it will stop completely.

Pharmaceutical compositions include compositions in which the active ingredient is contained in a therapeutically effective amount, that is, in an amount effective to achieve its intended purpose. The actual effective amount for a particular application will depend, among others, on the condition being treated. The determination of an effective amount is within the capabilities of those skilled in the art, especially in light of the detailed description of this invention.

For any compound described in this invention the therapeutically effective amount can be determined initially from cell culture assays. Target plasma concentrations will be those concentrations of active compound (s) that are capable of inhibiting cell growth or division. In preferred embodiments, cell activity is inhibited by at least 25%. Target plasma concentrations of active compound (s) that are capable of inducing at least about 50%, 75% or even 90% or more of cell activity inhibition are preferably present. The percentage of cell activity inhibition in the patient can be controlled to assess the adequacy of the plasma drug concentration achieved, and the dosage can be adjusted up or down to achieve the desired percentage of inhibition.

As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a circulating concentration that has been found to be effective in animals. The dosage in humans can be adjusted by controlling cell inhibition and adjusting the dosage up or down as described above.

A therapeutically effective dose can also be determined from human data for compounds that are known to show similar pharmacological activities. The applied dose can be adjusted based on the relative bioavailability and potency of the compound administered compared to the known compound.

Dose adjustment to achieve maximum efficacy in humans based on the procedures described above and other procedures as are well known in the art is within the capabilities of the person skilled in the art.

In the case of local administration the concentration in systemic circulation of administered compound will not be of particular importance. In such cases the compound is administered so that it reaches a concentration in the local area effective to achieve the intended result.

For use in the prophylaxis and / or treatment of diseases related to abnormal cell proliferation, a circulating concentration of compound administered from about 0.001 µM to 20 µM is preferred, with about 0.01 µM to 5 µM being preferred.

Doses for patients for oral administration of the compounds described in this invention typically range from about 1 mg / day to about 10,000 mg / day, more typically from about 10 mg / day to about 1,000 mg / day, and most typically from about 50 mg / day to about 500 mg / day. In terms of the patient's body weight, typical dosages range from about 0.01 to about 150 mg / kg / day, more typically from about 0.1 to about 15 mg / kg / day, and most typically from about 1 to about 10 mg / kg / day, for example 5 mg / kg / day or 3 mg / kg / day.

In at least some embodiments, patient doses that retard or inhibit tumor growth may be 1 µmol / kg / day or less. For example, patient doses may be 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or 0.1 µmol / kg / day or less (referred moles of the drug) of the drug or of a drug conjugate, such as an antibody-drug conjugate. Preferably, the drug or drug conjugate tumor growth when administered in the daily dosage amount for a period of at least five days. In at least some embodiments, the tumor is a human-type tumor in an SCID mouse. As an example, the SCID mouse can be a CB17.SCID mouse (available from Taconic, Germantown, NY).

For other modes of administration, the amount and dosage range can be adjusted individually by providing effective plasma levels of the administered compound for the particular clinical indication to be treated. For example, in one embodiment a compound according to the invention can be administered multiple times a day at relatively high concentrations. Alternatively it may be more desirable.

5 administering a compound of the invention in minimum effective concentrations and using a less frequent administration regimen. This will provide a therapeutic regimen that is commensurable with the severity of the individual disease.

By carrying out the indications provided in this invention, an effective therapeutic treatment regimen that does not cause substantial toxicity and is still completely effective for treating symptoms can be planned

10 clinicians demonstrated by the particular patient. This planning should involve the careful choice of the active compound by considering factors such as potency of the compound, relative bioavailability, body weight of the patient, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent. In general, the rest CBI presents the formula:

Where the substituents may be attached to the oxygen and nitrogen atoms, X is halo and R6 is H, substituted C1-6 alkyl or unsubstituted C1-6 alkyl, cyano or alkoxy. Preferably R6 is H, methyl or cyano. More preferably R6 is H. In addition X is preferably Cl or Br and more preferably X is Br-. In general, a unit may be attached to the amino substituent of the CBI moiety. Examples of suitable binding units include, but are not limited to,

wherein X1, Z, R4, R4 ', R5, and R5' are as defined above.

Examples of suitable binding units are illustrated and described in US patent applications serial numbers 10 / 160,972; 10 / 161,233; 10 / 161,234, 11 / 134,685, and 11 / 134,826; as well as in U.S. Patent No. 6,534,660. Suitable binding units within this formula also include units

25 connection with multiple condensation rings such as:

where Z 'is independently selected from O, S and NR8 where R8 is a member selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, and acyl.

R4 ", R4 '", R5 ", and R5'" are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, heterocycloalkyl unsubstituted, halogen, NO2, NR9R10, NC (O) R9, OC (O) NR9R10, OC (O) OR9, C (Q) R9, SR9, OR9, CR9 = NR10, and O (CH2) nNR11R11.

wherein R9 and R10 are independently selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, substituted aryl or unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl, substituted heterocycloalkyl or unsubstituted heterocycloalkyl, and substituted peptidyl or unsubstituted peptidyl, or in which R9 and R10 together with the nitrogen atom to which they are attached are optionally combined to form a substituted or unsubstituted heterocycloalkyl ring system having 4 to 6 members, optionally containing two or more heteroatoms, and

R11 and R11 are each independently H or substituted C1-6 alkyl or unsubstituted C1-6 alkyl.

R15 may be H, substituted alkyl or unsubstituted alkyl, or R15 and R4 'or R5' may be combined to form a ring (eg, a five or six membered ring).

An intermediate compound useful in the formation of CBI analogs CC-1065 has the formula (I):

wherein R1 and R2 are each independently H, alkyl, -C (O) OR ', -C (O) NR'R ", or a protecting group, wherein R' and R" are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl, and X is halogen.

In a conventional synthesis process for compound (I), the starting material is 1,3-dihydroxynaphthalene which is reacted with ammonia in a pressurized chamber (for example, a pump) to replace the hydroxy group at position 3 of the Naphthalene with an amine. Examples of this synthesis procedure can be found in U.S. Patent No. 6,534,600 and D.L. Boger et al., J. Org. Chem. 57, 2873-2876 (1992).

The amination reaction is followed by the addition of protecting groups to both the hydroxy moieties and the amine moieties.

While the amination reaction may have acceptable performance on a small scale, it may be difficult to increase the scale of the reaction due to the use of a pump to contain this pressurized reaction, which typically takes place at a pressure substantially greater than 1 atmosphere. (approximately 1.01 x 105 Pa) and in general at a pressure of at least 1.5 atmospheres (1.52 x 105 Pa). This synthesis procedure has been found to result in substantially lower yield when the scale is increased.

In contrast to conventional procedures, the starting material may be compound (II), as illustrated in the synthesis schemes of Figures 1 and 2:

wherein R3 is H or alkyl. Preferably, R3 is C1-5 alkyl and, more preferably, methyl. For example, 4-methoxy-2-naphthylamine is commercially available from Aldrich Chemical Company, Inc., Milwaukee, WI. It is relatively easy to hydrolyze the compound if R3 is alkyl, and add protective groups, R1 ′, to both hydroxy and amine moieties forming compound (III)

In some embodiments, as illustrated in Figures 1 and 2, it is desirable to provide different protecting groups in the amine and hydroxy functionalities. In accordance with the above, the initial protecting group, R1, can be released from one of these moieties (for example, from the hydroxy moiety) and replaced with a second different protecting group, R2 ’, giving the compound (IV)

A specific example of compound (IV) presents the formula:

By presenting different protecting groups in the hydroxyl and amine substituents, subsequent reaction steps can be facilitated where one of the protecting groups can be selectively removed while leaving the

15 another protecting group. Alternatively, different protecting groups may be initially added to the hydroxyl and amine substituents.

Schemes 1 and 2 (Figures 1 and 2) illustrate an embodiment of the remaining steps in the formation of the compound (I) from the compound (IV). These steps may include, for example, the formation of a ring using the amino group nitrogen. This can be achieved, for example, by alkylation of the aryl ring adjacent to the

20 nitrogen followed by a ring closure stage. In one embodiment the iodination of compound (IV) with Nyodosuccinimide results in a compound that can be rented using 1,3-dibromopropene or 1,3-dichloropropene. Ring closure can be carried out using tributyltin hydride in the presence of 2,2′-azobisisobutyronitrile (AIBN) giving the racemic CBI derivative, compound (V):

If desired, protective groups can be removed forming CBI. It will be understood that different reactants and catalysts can be used in these reaction steps. Examples can be found in Boger, Chemical Reviews, 97, 787-828 (1997).

The racemic CBI derivative can be separated using known enantiomer separation techniques including the use of chromatography procedures. A particularly useful technique is high performance liquid chromatography (HPLC) using a chiral column. For example, separation of such enantiomers has been carried out using a Chiralcel HPLC column and hexane / isopropanol eluent (99: 1) to give compound (I).

Specific examples of compound (I) include, but are not limited to,

Compound (I) can be used to form CBI analogs CC-1065 as described, for example, in US patent applications serial numbers 10 / 160,972; 10 / 161,233; 10 / 161,234, 11 / 134,685, and 1 1 / 134,826 of the applicant. For example, a binding unit can be added to the compound (I) by deprotection of the amino substituent and reacting a compound comprising the binding unit with the unprotected amine. Additional substituents may be added to the oxygen atom of the CBI compound by deprotection of the oxygen and reacted with appropriate reactant (s).

Examples

Without further elaboration, it is believed that one skilled in the art can, using the foregoing description, use the present invention to its fullest extent. The following preferred embodiments are therefore constructed as merely illustrative.

In the foregoing and in the following examples, all temperatures are indicated uncorrected in degrees Celsius and all parts and percentages are by weight, unless otherwise indicated.

Example 1 - Scheme 1 (Figure 1)

Synthesis of N- (tert-butyloxycarboni) -4-O- (tert-butyloxycarbony) -2-naphthylamine (2)

A solution of 4-methoxy-2-naphthylamine (230 mg, 1.33 mmol) in glacial acetic acid (9.6 ml) and hydrobromic acid in water (16 ml, 48%) in N2 was refluxed for 4 h . A small amount of sample (0.1 ml) was diluted with ethyl acetate (0.5 ml), and then water (0.5 ml) and TEA (0.1 ml) were added. TLC (DCM / methanol 20: 1) of the organic layer showed no starting material and a much lower new spot (Rf = 0.1). The solvent was removed under reduced pressure and the product was dried under vacuum giving intermediate 4-hydroxy-2-naphthylamine which was used for the next step without any purification. To a solution of 4-hydroxy-2-naphthylamine in dioxane (10 ml) TEA (1 ml) and di-tert-butyl dicarbonate (1,149 g, 5.27 mmol) was added. The reaction mixture was refluxed in N2 for 4 h. TLC (hexane / ethyl acetate 4: 1) showed no starting material and a new upper spot (Rf = 0.55). The reaction mixture was diluted with ethyl acetate (50 ml) and washed with water. The aqueous layer was extracted with ethyl acetate (2 x 50 ml) and the organic extracts were combined and washed with brine. The organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give N- (tert-butyloxycarbonyl) -4-O- (tert-butoxycarbonyl) -2-naphthylamine (2.80% yield) as oil.

Synthesis of compound 3

To a solution of compound 2 in acetone (10 ml) solution of NaOH in water (10 ml, 1 M) was added. The reaction mixture was stirred at room temperature overnight. TLC (hexane / ethyl acetate 4: 1) showed no starting material and a new lower spot. The reaction mixture was extracted with ethyl acetate (50 ml) and washed with water. The aqueous layer was extracted with ethyl acetate (2 x 50 ml) and the organic extracts were washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified on a 10 g silica gel column with 0-20% ethyl acetate in hexane to give compound 3 (181 mg, 53%) as an oil.

Synthesis of compound 4:

A solution of compound 3 (5 g, 19.3 mmol) in anhydrous DMF (50 ml) was treated under a nitrogen atmosphere with benzyl bromide (4 g, 23.1 mol), potassium carbonate (3.7 g, 27 moles) and tetrabutylammonium iodide (70 mg, 0.01 mmol). The reaction mixture was stirred at room temperature for 8 h. The reaction mixture was concentrated under reduced pressure. Chromatographic separation (4 x 10 cm of SiO2, gradient elution of 10-20% EtOAchexane) gave pure compound 4 (5.48 g, 83%) as a cream powder. 1H NMR (CDCl3, 400 MHz, ppm) 8.22 (d, 1H J = 8.1 Hz, C5-H), 7.68 (d, 1H, J = 8.2 Hz5 C8-H), 7, 3-7.5 (m, 8H, Cl-H, C6-H, C7-H, CH2C6H5), 7.06 (d, 1H, J = U Hz, C3-H), 6.62 (sa, 1H , NH), 5.23 (S, 2H, OCH2 (C6H5), 1.55 (s, 9H, OC (CH3) 3).

Synthesis of compound 5:

Compound 4 (13 g, 0.0372 mol) and THF (300 ml) were combined in a 1000 ml round bottom flask equipped with a stir bar and a rubber septum. The light yellow solution was cooled to -20 ° C with a dry ice bath in a nitrogen atmosphere. P-Toluenesulfonic acid (0.10 g, 0.0005 mol) was added to the reaction and the solution was stirred for 10 minutes. N-iodosuccinimide (10 g, 0.0446 mol) was dissolved in THF (50 ml) and added to the reaction by cannula (approximately 1 hour). The solution was stirred in an ice bath for 2 hours and turned brownish. The solution was then removed from the ice bath and allowed to warm to room temperature under nitrogen for 1.5 hours. TLC (hexane / DCM 2: 1) showed no starting material and a new upper stain. The reaction was stopped with saturated NaHCO3 (200 ml) and a white solid formed. After stirring the solution for 10 minutes, EtOAc (200 ml) and water (100 ml) were added to the reaction. The aqueous layer was extracted with EtOAc (2 x 100 ml) and the organic extracts were combined and extracted with brine (100 ml). The organic extracts were dried over MgSO4, filtered, and concentrated in vacuo to a dark reddish-brown solid. The solid was purified by column chromatography using hexane / DCM 2: 1 as eluent to give compound 5 (14 g, 79%) as a brown solid.

Synthesis of compound 6:

Compound 5 (22.5 g, 0.0473 mol) and anhydrous DMF (250 ml) were combined in a 500 ml round bottom flask equipped with a stir bar and nitrogen inlet. The yellowish orange solution was cooled to 0 ° C with an ice / salt bath in a nitrogen atmosphere. NaH (60%, 5.6 g, 0.146 mol) was added to the reaction once. The solution became cloudy and a gas formed. The reaction was stirred in an ice bath for 15 minutes and then the ice bath was removed and the solution was stirred for another 15 minutes, cis / trans-1,3-dibromopropene (14 ml, 0.14 mol) was added to the portion reaction by syringe. The reaction was stirred under nitrogen at room temperature for 1 hour and turned cloudy brown. The temperature increased to 40 ° C. The reaction mixture was allowed to cool to room temperature. TLC (hexane / EtOAc 4: 1) showed no starting material and a new lower stain. The reaction was stopped with water (500 ml). The aqueous layer was extracted with EtOAc (4 x 100 ml) and the organic extracts were washed with brine (2 x 75 ml). The organic extracts were dried over MgSO4, filtered and concentrated in vacuo to a brown oil. The product was purified by column chromatography using DCM / hexane 1: 1 as eluent to give compound 6 (25 g, 89%) as a brown oil.

Synthesis of compound 7:

It was combined in a round bottom flask of three 1000 ml mouths equipped with stir bar, temperature probe, reflux condenser and nitrogen inlet compound 6 (25 g, 0.0421 mol), toluene (500 ml), 2, 2'-azobis (2-methylpropionitrile) (0.15 g, 0.0009 mol) and tributyltin hydride (3.4 ml, 0.0126 mol) [by syringe]. Nitrogen was bubbled through the solution for 15 minutes and then the reaction was heated to 80 ° C under nitrogen. After heating at 80 ° C for 15 minutes, tributyltin hydride (3.4 ml, 0.0126 mol) was added to the reaction by syringe. After heating for another 15 minutes, tributyltin hydride (3.4 ml, 0.0126 mol) was added to the reaction by syringe. After another 15 minutes, tributyltin hydride (3.4 ml, 0.0126 mol) was added to the reaction by syringe. The total amount of tributyltin hydride added was 13.6 ml, 0.0505 mol. The reaction was heated at 80 ° C for 30 minutes and then allowed to cool to room temperature. TLC (10% EtOAc / hexane) showed starting material and a new upper stain. The solution was concentrated in vacuo to a yellow solid. The solid was purified by column chromatography using 1: 1 dichloromethane / hexane as eluent to give a yellow solid. The solid was recrystallized from hexane (200 ml, 45 ° C for 30 minutes, cooled in a refrigerator for 2 hours, collected by filtration, dried under vacuum) to give compound 7 (11.70 g, 59% yield) as a pale yellow solid. NMR (1H, CDCl3, 400 MHz): � 1.61 (9H, s, C (CH3) 3); 3.30 (1H, t, J = 26 Hz, CH-CH2-N); 3.82 (1H, d, J = 26 Hz, Br-CH2-CH); 4.04 (1H, d, J = 19 Hz, Br-CH2-CH) 4.14 (1H, t, J = 26 Hz, CH-CH2-N); 4.21 (1H, m, CH2-CH-CH2); 5.26 (2H, s, OCH2-C6H5); 7.3-7.55 (8H, m, O-CH2-C6H5, C10H5); 7.63 (1H, d, J = 21 Hz, C10H5); 8.3 (1H, d, J = 21 Hz, C10H5).

Resolution of compound 7:

The racemic compound 7 was dissolved in DCM (50 mg, 1 ml). The solution was then diluted with hexane (9 ml). The solution was then loaded onto a Chiralcel OD preparative column (10 micrometers, 20 x 250 mm) and separated using hexane / isopropanol (99: 1, 15 ml / min). The first enantiomer (7a) elutes from 10 to 15 minutes and the second enantiomer (7b) elutes from 17.5 to 25 minutes. The analytical column (Chiralcel OD, 0.46 x 25 cm, 20 microns) gives a retention time of 7.71 minutes for compound 7a and 12.9 minutes for compound 7b (hexane / IPA 99: 1, 1 ml / min, 15 minute trial). 1H NMR (CDCl3, 400 MHz): D 1.61 (9H, s, C- (CH3) 3); 3.30 (1H, t, J = 26 Hz, CH-CH2-N); 3.82 (1H, d, J = 26 Hz, Br-CH2-CH); 4.04 (1H, d, J = 19 Hz, Br-CH2-CH) 4.14 (1H, t, J = 26 Hz, CH-CH2-N); 4.21 (1H, m, CH2-CH-CH2); 5.26 (2H, s, O-CH2-C6H5); 7.3-7.55 (8H, m, O-CH2-C6H5, C10H5); 7.63 (1H, d, J = 21 Hz, C10H5); 8.3 (1H, d, J = 21 Hz, C10H5).

Example 2 - Scheme 2 (Fig. 2)

The synthesis procedure is as described above in Example 1 by the synthesis of the compound.

5.

Synthesis of compound 8:

It was combined in a 250 ml round bottom flask equipped with stir bar and nitrogen inlet compound 5 (8.4 g, 0.0177 mol) and anhydrous DMF (125 ml). The yellowish orange solution was cooled to 0 ° C with an ice / salt bath in a nitrogen atmosphere. NaH (60%, 2.22 g, 0.0554 mol) was added to the reaction once. The solution became cloudy and a gas formed. The reaction was stirred in an ice bath for 15 minutes and then the ice bath was removed and the solution was stirred for another 15 minutes, cis / trans-1,3-dichloropropene (5.3 ml, 0.0571 mol) was added to the reaction in portions by syringe. The reaction was stirred under nitrogen at room temperature for 3 hours and turned cloudy brown. TLC (hexane / EtOAc 4: 1) showed no starting material and a new lower spot. The reaction was stopped with water (250 ml). The aqueous layer was extracted with EtOAc (3 x 100 ml) and the organic extracts were washed with brine (2 x 50 ml). The organic extracts were dried over MgSO4, filtered and concentrated in vacuo to a brown solid. The product was purified by column chromatography using DCM / hexane 1: 1 as eluent to give (E / Z) -terc-butyl 4- (benzyloxy) -1-iodophthalen-2-yl (3-chloroalyl) carbamate (8) (9 g, 93%) as a yellow oil.

Synthesis of compound 9:

The compound 8 (9 g, 0.0164 mol), toluene (200 ml), 2 was combined in a 500 ml round bottom flask equipped with a stir bar, temperature probe, reflux condenser and nitrogen inlet. , 2'-azobis (2-methylpropionitrile) (0.15 g, 0.0009 mol) and tributyltin hydride (1.5 ml, 0.0056 mol) [by syringe]. Nitrogen was bubbled through the solution for 15 minutes and then the reaction was heated to 80 ° C under nitrogen. After heating at 80 ° C for 15 minutes, tributyltin hydride (1.5 ml, 0.0056 mol) was added to the reaction by syringe. After heating for another 15 minutes, tributyltin hydride (1.5 ml, 0.0056 mol) was added to the reaction by syringe. After another 15 minutes, tributyltin hydride (1.0 ml, 0.0037 mol) was added to the reaction by syringe. The total amount of tributyltin hydride added was 5.5 ml, 0.0204 mol. The reaction was heated at 80 ° C for 30 minutes and then allowed to cool to room temperature. TLC (10% EtOAc / hexane) showed no starting material and a new upper stain. The solution was concentrated in vacuo to give a yellow oil. The oil was purified by column chromatography using 100% hexane as eluent to 5% EtOAc / hexane to 10% EtOAc / hexane to give a pale yellow solid. The solid was recrystallized from hexane (100 ml, 45 ° C for 30 minutes, cooled in a refrigerator for 2 hours, collected by filtration, dried under vacuum) to give compound 9 (4.16 g, 60% yield) as a white solid

Resolution of compound 9:

The racemic compound 9 was dissolved in DCM (50 mg, 1 ml). The solution was then diluted with hexane (9 ml). The solution was then loaded onto a Chiralcel OD preparative column (10 micrometers, 20 x 250 mm) and separated using hexane / isopropanol (99: 1, 15 ml / min). The first enantiomer (9a) elutes from 11.5 to 15 minutes and the second enantiomers (9b) elute from 17.5 to 25 minutes. The analytical column (Chiralcel OD, 0.46 x 25 cm, 20 micrometers) gives a retention time of 6.5 minutes for compound 9a and 10.6 minutes for compound 9b

(99: 1 hexane / IPA, 1 ml / min, 15 minutes of test). 1H NMR (CDCl3, 400 MHz): 1.61 d (9H, s, C- (CH3) 3); 3.44 (1H, t, J = 25 Hz, CH-CH2-N); 3.9-4.0 (2H, m, Cl-CH2-CH); 4.12 (1H, t, J = 26 Hz, CH-CH2-N); 4.25 (1H, m, CH2-CH-CH2); 5.26 (2H, s, 0-CH2-C6H5); 7.2-7.5 (8H, m, O-CH2-C6H5, C10H5); 7.63 (1H, d, J = 21 Hz, C10H5); 8.3 (1H, d, J = 21 Hz, C10H5).

Example 3 - Scheme 3 (Figure 3)

The synthesis procedure is as described above in example 1 with the synthesis of compound 3.

Synthesis of compound 10:

A solution of tert-butyl 4-hydroxynaphthalene-2-ylcarbamate (3 mg (500 mg, 2.89 mmol), 4-methyl-1-piperazinecarbonyl chloride hydrochloride (858 mg, 4.34 mmol) was stirred, anhydrous pyridine (4.98 ml, 57.8 mmol), and allyl alcohol (4.98 ml, 73.2 mmol) in anhydrous DCM (20 ml) at room temperature overnight. TLC (DCM / MeOH 9: 1) showed no starting material and a much lower stain. The reaction mixture was interrupted with water. The aqueous layer was extracted with EtOAc (3 x 50 ml) and the organic extracts were washed with brine (2x 50 ml). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to brown oil. The crude product was purified by column chromatography with 1-5% methanol in DCM to give compound 10 (602 mg, 82%) as a yellow solid. 1H NMR (DMSO-d6) 5 9.68 (s, 1H), 7.91 (s, 1H), 7.80 (d, 1H), 7.69 (s, 1H), 7.47 (t, 1H), 7.42 (s, 1H), 7.39 (t, IH), 3.78 (s, 2H), 3.42 (s, 2H), 2.44 (s, 2H), 2, 39 (s, 2H), 2.21 (s, 3H), 1.50 (s, 9H).

Synthesis of compound 11:

Compound 10 (82 mg, 0.21 mmol), p-toluenesulfonic acid (10 mg, 0.05 mmol), and N-iodosuccinimide (96 mg, 0.43 mmol) in anhydrous THF (5 ml) was stirred at temperature overnight atmosphere. TLC (DCM / MeOH 9: 1) showed a small amount of starting material and a superior stain. The reaction mixture was quenched with saturated NaHCO3 (10 ml). After stirring at room temperature for 10 minutes, the reaction mixture was extracted with EtOAc (3 x 20 ml) and the organic extracts were washed with brine (2 x 20 ml). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to brown oil. The crude product was purified by column chromatography with 1-5% methanol in DCM to give compound 11 (52 mg, 48%) as yellow oil. 1H NMR (DMSO-d6) 5 8.81 (s, 1H), 8.14 (d, 1H), 7.79 (d, 1H), 7.65 (t, 1 H), 7.58 (t , 1H), 7.45 (t, 1H), 3.78 (s, 2H), 3.42 (s, 2H), 2.44 (s, 2H), 2.39 (s, 2H), 2 , 21 (s, 3H), 1.50 (s, 9H).

Synthesis of compound 12:

A solution of compound 11 (102 mg, 0.2 mmol) in anhydrous DMF (5 ml) was cooled in an ice bath. Sodium hydride (60% in mineral oil, 32 mg, 0.8 mmol) was added to the reaction. The reaction mixture was stirred at 0 ° C for 15 minutes and at room temperature for 15 minutes. Cis / trans-1,3-dichloroprepene (83.36 µL, 0., mmol) was added to the reaction. The reaction mixture was stirred at room temperature for 1 hour. TLC (DCM / MeOH 9: 1) showed no starting material. The reaction mixture was interrupted with water. The aqueous layer was extracted with EtOAc (3 x 10 ml) and the organic extracts were washed with brine (2 x 10 ml). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to brown oil. The crude product was purified by column chromatography with 1-5% methanol in DCM to give compound 12 (82 mg, 70%) as a yellow solid.

1H NMR (DMSO-d6) 5 8.20 (d, 1H), 7.82 (d, 1 H), 7.62 (m, 2H), 7.38 (d, 1H), 6.38 (m , 1H) 5 6.18 (m, 1H), 3.98-4.46 (dd, 2H), 3.78 (s, 2H), 3.42 (s, 2H), 2.44 (s, 2H), 2.39 (s, 2H), 2.21 (s, 3H), 1.50 (s, 9H).

Synthesis of compound 13:

It was bubbled in a solution of compound 12 (82 mg, 0.14 mmol) in anhydrous toluene (3 ml), dry nitrogen during

5 15 minutes. Tributyltin hydride (47.1 µL, 0.18 mmol) and 2,2'-azobisisobutyronitrile (10 mg, 0.06 mmol) were added to the reaction. The reaction mixture was heated at 80 ° C for 15 minutes under nitrogen. The TLC (DCM / MeOH 9: 1) showed new blue spot and no starting material. The reaction mixture was concentrated in vacuo to give yellow oil. The crude product was purified by column chromatography with 1-5% methanol in DCM to give compound 13 (52 mg, 82%) as a white solid. 1H NMR (DMSO-d6) 5 7.92 (d, 1H), 7.83 (m, 1H),

10 7.78 (d, 1 H), 7.58 (t, 1H), 7.42 (t, 1H), 4.20 (m, 2H), 4.04 (m, 2H), 3.92 (m, 1H), 3.78 (s, 2H), 3.42 (s, 2H), 2.44 (s, 2H), 2.39 (s, 2H), 2.21 (s, 3H) , 1.50 (s, 9H).

Resolution of compound 13:

The racemic compound 13 is dissolved in methanol. The solution is then loaded into a CHIRALPAK AD preparative column (20 micrometers 20 x 250 mm) and separated using methanol (15 ml / min). The first enantiomer

15 (13a) elutes from 5.1 minutes and the second enantiomer (13b) elutes from 7.1 min. 1H NMR (DMSO-d6) 5 7.92 (d, 1H), 7.83 (m, 1H), 7.78 (d, 1 H), 7.58 (t, 1H), 7.42 (t , 1H), 4.20 (m, 2H), 4.04 (m, 2H), 3.92 (m, 1H), 3.78 (s, 2H), 3.42 (s, 2H), 2 , 44 (s, 2H), 2.39 (s, 2H), 2.21 (s, 3H), 1.50 (s, 9H).

Example 4 - Synthesis of CBI analogue CC-1065

20 Synthesis of compound (14). A solution of compound 9b (100 mg, 0.24 mmol) and 10% Pd-C (35 mg) in MeOH / CH2Cl2 (1/2, 10 ml) was degassed under vacuum for 40 s. The resulting mixture was placed under a hydrogen atmosphere and stirred at 25 ° C for 7 h. The reaction mixture was filtered through Celite (washed with CH2CI2). The solvent was removed in vacuo. Chromatography on silica gel eluted with EtOAc / Hex (2/8) gave compound 14 (77

25 mg, 98%). 1H NMR DMSOd6) 5 10.36 (s, 1H), 8.04 (d, 1H, J = 8.2 Hz), 7.72 (d, 1H, J = 8.2 Hz), 7.61 ( sa, 1H), 7.45 (t, 1H, J = 8.4 Hz), 7.261 (t, 1H, J = 8.4 Hz), 4.06 (m, 4H), 3.73 (m, 1H), 1.52 (s, 9H).

Synthesis of the compound (16). A solution of compound 14 (35 mg, 0.1 mmol) in 4M HCl-EtOAc (5 mL) was stirred at 25 ° C in Ar for 30 minutes. The solvent was removed in vacuo. 5-Acetylindon-2-carboxylic acid (24.4 mg, 0.12 mmol) was added to the residue. A solution of EDC (22.9 mg, 0.12 mmol) in DMF (3 ml) was added and the reaction mixture was stirred at 25 ° C for 5 h. The solvent was removed. The crude product was subjected to chromatography in

silica gel eluting with 10% MeOH in CH2Cl2 to give compound 16 (40.7 mg, 93%). 1H NMR DMSOd6) 512.13 (s, 1H), 10.47 (s, 1H), 8.45 (s, 1H), 8.10 (d, 1H, J = 8.4 Hz), 7.96 (sa, 1H), 7.85 (d, 2H, J = 8.4 Hz), 7.54 (d, 1H, J = 8.4 Hz), 7.51 (t, 1H, J = 8, 2 Hz), 7.36 (t, 1H, J = 7.6), 7.35 (s, 1H), 4.81 (t, 1H, 11.2 Hz), 4.54 (dd, 1H, 8.8 Hz), 4.23 (m, 1H), 4.01 (dd, 1H, J = 10.2 Hz), 3.86 (dd, 1H, J = 10.7 Hz), 2.61 (s, 3H).

Synthesis of the compound (17). 4-Methyl-1-piperazinecarbonyl chloride hydrochloride (19.9 mg, 0.1 mmol) was added to a solution of compound 16 (20 mg, 0.05 mmol) and anhydrous pyridine (25 µm, 0.3 mmol) in 3% allyl alcohol in dry methylene chloride (4 ml) and the mixture was stirred for 16 h. Purification of the crude product on silica gel gave compound 17 (23.6 mg, 91%). 1H NMR DMSOd6) 5 12.03 (s, 1H), 8.41 (s, 1H), 8.21 (s, 1H), 8.01 (d, 1H, J = 8.4 Hz), 7, 88 (d, 1H, J = 8.4 Hz), 7.82 (dd, 1H, J = 8.4 Hz), 7.58 (t, 1H, J = 8.1 Hz), 7.51 ( d, 1H, J = 8.4 Hz) 5 7.46 (t, 1H, J = 7.6 Hz), 7.37 (s, 1H), 4.86 (t, 1H, J = 10.8 Hz), 4.57 (dd, 1H, J = 10.8 Hz), 4.38 (in, 1H), 4.06 (dd, 1H, J = I 0.8 Hz), 3.86 (dd , 1H, J = I 1 Hz), 3.41 (br, 4H), 3.29 (br, 4H), 2.82 (s, 3H), 2.57 (s, 3H).

Synthesis of the compound (19). A solution of compound 17 (13 mg, 24 umol) and connector 18 (16.9 mg, 31 umol) in 5% acetic acid in dry methylene chloride (1 ml) was stirred for 30 min at 25 ° C. the solvent completely under vacuum and purified by HPLC (SymmetryPrep C18 column, 7 µm, 19 x 150 mm) to give compound 19 (18.5 mg, 81%). MS: calculated for C48H57ClN8On (M + H) m / z 958.38, found 958.10.

Example 5: proliferation assays

The biological activity of the cytotoxic compounds of the invention can be tested using the well-defined 3H-thymidine proliferation assay. This is a convenient procedure for the quantification of cell proliferation, since it evaluates DNA synthesis by measuring the incorporation of radiologically exogenously labeled 3H-thymidine. This assay is highly reproducible and can accommodate a large number of compounds.

To carry out the assay, promyelocytic leukemia cells, HL-60, are grown in RPMI medium containing 10% heat-inactivated fetal bovine serum (FCS). On the day of the study, the cells are collected, washed and resuspended at a concentration of 0.5 x 10 6 cells / ml in RPMI containing 10% FCS. 100 µl of cell suspension is added to 96-well plates. Serial dilutions (3-fold increments) of dexorubicin (as a positive control) are added or test compounds are prepared and 100 µl of compounds are added per well. Finally, 10 µl of a 3H-thymidine 100 µC / ml per well is added and the plates are incubated for 24 hours. Plates are grown using a 96-well harvester (Packard Instruments) and counted in a Packard Top Count counter. Logistic parameter curves are adjusted with the incorporation of 3H-thymidine as a function of drug molarity using Prism software to determine IC50 values.

CBI analogs CC-1065 (for example, compound 19 of example 4) generally have an IC50 value in the previous test of about 1 pM to about 100 nM, preferably about 10 pM to about 10 nM.

Example 6: conjugation of drug molecules with antibodies

This example describes the reaction conditions and methodologies for the conjugation of a drug molecule of the invention (optionally including other groups, such as spacers, reactive functional groups and the like) with an antibody as a targeting agent. The conditions and methodologies are intended to be exemplary only and not limiting. Other approaches to the conjugation of drug molecules with antibodies are known in the art.

The conjugation process described in this invention is based on the introduction of free thiol groups with the antibody by reaction of lysines of the antibody with 2-iminothiolanes, followed by reaction of the drug binding molecule with an active maleimide group. Initially the antibody to be conjugated is buffered interchangeably in 0.1 M phosphate buffer pH 8.0 containing 50 mM NaCl, 2 mM DTPA, pH 8.0 and concentrated at 5-10 mg / ml. Thiolation is achieved by adding 2-iminothiolane to the antibody. The amount of 2iminothiolane to be added is determined in preliminary experiments and varies from one antibody to another antibody. In preliminary experiments a titration of increasing amounts of 2-iminothilane is added to the antibody, and after incubation with the antibody for one hour at room temperature the antibody is desalted in 50 mM HEPES buffer pH 6.0 using a Sephadex G- column. 25 and the number of thiol groups introduced is rapidly determined by reaction with dithiodipyridine (DTDP). The reaction of thiol groups with DTDP results in the release of thiopyridine which is controlled at 324 nm. Samples can be used at a protein concentration of 0.5-1.0 mg / ml. The absorbance at 280 nm is used to determine exactly the protein concentration in the samples, and then an aliquot of each sample (0.9 ml) is incubated with 0.1 ml of DTDP (5 mM stock solution in ethanol) for 10 minutes at room temperature. They are also incubated with these white buffer samples alone plus DTDP. After 10 minutes the absorbance at 324 nm is measured and the number of thiols present is quantified using a thiopyridine extinction coefficient of 19800 M-1.

Typically, a thiolation level of three thiol groups per antibody is desired. For example, with a given antibody this can be achieved by adding a 15-fold molar excess of 2-iminothiolane followed by incubation at room temperature for 1 hour. The antibody to be conjugated is therefore incubated with 2-iminothiolane at the desired molar ratio and then desalted in conjugation buffer (50 mM HEPES buffer pH 6.0 containing 5 mM glycine, 3% glycerol and DTPA 2 mM). The thiolated material is kept on ice while the number of thiols introduced is quantified as described above.

After verification of the number of thiols introduced the drug molecule containing an active maleimide group

5 (for example, compound 15 of example 3) is added in a molar excess of 3 times per thiol. The conjugation reaction is carried out in conjugation buffer which also contains a final concentration of 5% ethylene glycol dimethyl ether (or a suitable alternative solvent). Usually, the stock drug solution dissolves 90% ethylene glycol dimethyl ether, 10% dimethylsulfoxide. For addition to the antibody, the stock solution is added directly to the thiolated antibody, which has enough ethylene glycol dimethyl ether added to carry the

The final concentration is up to 5%, or it is prediluted in conjugation buffer containing a final concentration of 10% ethylene glycol dimethyl ether, followed by the addition of an equal volume of thiolated antibody.

The conjugation reaction is incubated at room temperature for 2 hours with mixing. After incubation, the reaction mixture is centrifuged at 14000 rpm for 15 minutes and the pH can be adjusted to 7.2 if purification is not immediate. Conjugate purification can be achieved by chromatography using a number of procedures. The conjugate can be purified using size exclusion chromatography on a Sephacryl S200 column pre-equilibrated with 50 mM HEPES buffer pH 7.2 containing 5 mM glycine, 50 mM NaCl and 3% glycerol. Chromatography can be carried out with a linear flow rate of 28 cm / h. Fractions containing conjugate can be collected, collected and concentrated. Alternative purification can be achieved by ion exchange chromatography. Conditions vary from one antibody to another and need to be

20 optimized in each case. For example, the antibody-drug conjugate reaction mixture can be applied to a SP-Sepharose column pre-equilibrated in 50 mM HEPES, 5 mM glycine, 3% glycerol, pH 6.0. The antibody conjugate can be eluted using a gradient of 0-1 M NaCl in equilibration buffer. The fractions containing the conjugate can be pooled, the pH can be adjusted to 7.2 and the concentrated sample as required.

The preceding examples can be repeated with similar success by replacing the reactants and / or

25 operating conditions described genetically or specifically of this invention by those used in the preceding examples.

From the foregoing description, a person skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt them to various uses and conditions.

Claims (8)

1.- A manufacturing process of a compound (I) or a salt thereof, 5 wherein R1 and R2 are each independently H, substituted alkyl or unsubstituted alkyl, -C (O) OR ', -C (O) NR'R ", or a protecting group, wherein R' and R "are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl, R6 is H, substituted C1-6 alkyl or alkyl C1-6 unsubstituted, cyano or alkoxy; and X is halogen, the process comprising: 10 the addition of protective groups R1 'and R2' to a compound (II) (II) to form a compound (III) (III) in which R3 is substituted alkyl or unsubstituted alkyl; and generating a five-membered ring comprising the amino nitrogen of compound (III) by alkylation of the aryl ring adjacent to nitrogen followed by ring closure. twenty 2. The process of claim 1, wherein R3 is methyl. 3. The method of claim 1, wherein X is Cl or Br. 4. The method of claim 1, wherein X is Br. 5. The method of claim 1, wherein R1 ’and R2’ are different protecting groups. The method of claim 5, wherein the addition of R1 ’and R2’ comprises the addition of R1 ’to the compound (II) to form the compound (III ’) and replace the protective group R1 ’in the hydroxyl substituent with the protective group R2’. 7. The process of claim 5, wherein R1 ’is tert-butyloxycarbonyl. 8. The method of claim 7, wherein R2 ’is -CH2Ph. The method of claim 1, wherein R1 ’and R2’ are the same. 10. The method of claim 1, which comprises replacing R1 ’and R2’ with hydrogen after generation of the five-member ring. 11. The method of claim 1, wherein R1 ’and R1 are the same and R2’ and R2 are the same. 12. The method of claim 1, wherein the five-member ring generation comprises the Iodination of a carbon adjacent to the amino substituent of compound (III) followed by alkylation using 1.3 dihalopropene 13.- A manufacturing process for a CBI analogue CC-1065, or a pharmaceutically acceptable salt of the same, which presents the following formula: in which X is halo; X1 and Z are each independently selected from O, S and NR8, wherein R8 is a member selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, and acyl; R4, R4 ', R5 and R5 are members independently selected from the group consisting of H, substituted alkyl, 20 unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO2, NR9R10, NC (O) R9, OC (O) NR9R10, OC (O) OR9, C (O) R9, SR9, OR9 and CR9 = NR10, wherein R9 and R10 are independently selected from H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl, substituted aryl or unsubstituted aryl, heteroaryl 25 substituted or unsubstituted heteroaryl, substituted heterocycloalkyl or unsubstituted heterocycloalkyl, and substituted peptidyl or unsubstituted peptidyl, or those that R9 and R10 together with the nitrogen atom to which they are attached are optionally combined to form a substituted or non-substituted heterocycloalkyl ring system substituted with 4 to 6 members, optionally containing two or more heteroatoms, and R11 and R11 'are each independently H or substituted C1-6 alkyl or unsubstituted C1-6 alkyl; R6 is H, substituted C1-6 alkyl or unsubstituted C1-6 alkyl, cyano, or alkoxy, and R7 is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates , triphosphates, acyl, C (O) R12R13, C (Q) OR12, C (O) NR12R13, P (O) (OR12) 2, C (O) CHR12R13, SR12 and SiR12R13R14, in which R12, R13, and R14 are independently selected members of H, substituted alkyl or unsubstituted alkyl, substituted heteroalkyl or unsubstituted heteroalkyl and substituted aryl or unsubstituted aryl, or in which R12 and R13 together with the nitrogen atom or carbon to which they are attached are optionally combined to form a substituted or unsubstituted heterocycloalkyl ring system having 4 to 6 members, optionally containing two or more heteroatoms; Understanding the procedure: the addition of protective groups R1 'and R2' to a compound (II) to form a compound (III) wherein R3 is substituted alkyl or unsubstituted alkyl; generating a five-membered ring comprising the amino nitrogen of compound (III) by alkylation of the aryl ring adjacent to the nitrogen followed by ring closure; and adding a binding unit to compound (III) by removing the protective group R1 ’and joining the rest: to the amino substituent of compound (III), in which indicates the point at which the residue is attached to the amino substituent. The method of claim 13, wherein the generation of the five-membered ring comprises the iodination of a carbon adjacent to the amino substituent of the compound (III) followed by alkylation using 1,3-dihalopropene before the generation of the ring. Five members 15. The method of claim 13, wherein R6 is H. 16. The method of claim 13 wherein R1 ’and R2’ protecting groups are selected from: BOC, FMOC, 25 2-Trimethylsilylethoxycarbonyl, allyloxycarbonyl, 4-methyl-1-piperazincarbonyl, 1-methyl-1- (4-biphenylyl) ethoxycarbonyl, diphenyloxycarbonyl, benzyl, t-butyl, tetrahydropyran, trimethylsilyl, t-butyldimethylsilyl, butyl, diphenyl-butylsilyl, butyl, diphenyl-butyl, butyl, diphenyl-butyl, butyl, diphenyl-butylsilyl 2,2,2-trichloroethyloxycarbonyl, diisopropylmethylxycarbonyl, vinyloxycarbonyl, methoxybenzyloxycarbonyl, nitrobenzyloxycarbonyl, cyclohexyloxycarbonyl, cyclopentyloxycarbonyl, formyl, acetyl, trihaloacetyl, benzoyl, nitrophenylacetyl, 2-nitrobenzylthio, cyl, nitrobenzeneside Figure 1 Figure 2 Figure 3